Den PHI och HoloMonitorförknippade danskättade forskaren Louise Sternbæk har genomfört studier som kommer förära henne titeln Dr och naturligtvis en åtråvärd doktorshatt.Om ca 2 veckor kommer Louise försvara (disputation) sin doktorsavhandling,därefter öppnas dörrarna till den prestigefyllda forskningen som ansamlar kollegor världen över. Bloggen vill redan här gratulera Louise till sin nya position. Grattis !
Men till doktorsavhandlingen som handlar om studier kring cancer och hur behandla mer effektivt än dagens teknik. Det genom MIP`s och det efterlängtade projektet GlycoImaging.
LOUISE STERNBÆK
Novel tools for detection, imaging, and analysis (PDF)
2022-11-25
Abstract
Sialic acid (SA) plays a crucial role in many biological processes. Cell surface
SA expression is usually analyzed with antibodies or lectins; however, they are
costly and with poor stability. We have used a molecular imprinting technique to
synthesize an alternative SA receptor – SA molecularly imprinted polymers
(SA-MIPs) with an embedded fluorophore for fluorescent detection of the
SA-MIPs. The binding behavior and specificity of SA-MIPs were verified by
using lectins and SA conjugates on cancer cell lines, showing that SA-MIPs can
be used as an effective tool for SA expression analysis of cancer cells. Digital
holographic cytometry (DHC) is a non-phototoxic quantitative phase imaging
technique that facilitates the monitoring of living cells over time. We have
demonstrated the potential of DHC by mapping cellular parameters, such as cell
number, area, thickness, and volume. In addition, cellular parameters possibly
depending on sialylation, were evaluated using DHC. Furthermore, the uptake
over time of SA-MIPs by macrophages was investigated for any inflammatory
and/or cytotoxic responses when administered to phagocytosing cells. Our results
indicate that SA-MIPs caused low induction and sparse secretion of inflammatory
cytokines, and that reduced cell proliferation was not due to cytotoxicity, but to
attenuated cell cycles. These results suggest that SA-MIPs will contribute to the
further understanding of cancer cell behavior and can be an asset for in vivo
studies.
Det vimlar av referenser till HoloMonitor så jag kommer enbart ha med få av dessa. Det för att inte skymma strålkastarljuset på Loise och hennes gedigna arbete. Gå in på länken och läs hela hennes doktorsavhandling.
Inledning
Digital holografisk cytometri är en mikroskopteknik som kan analysera levande
celler i en för cellerna optimal miljö och detta kan ske under en längre tid, såsom under 48 timmar.
Informationen som erhålls är exempelvis cellantal, cellernas
rörelsemönster och cellernas form med avseende på yta, volym och tjocklek.
Med
hjälp av mikroskopet och dess tillhörande program kunde vi under 48 timmar
följa cellerna en och en.
För att kunna använda plastpartiklarna på djur eller på människor, är det viktigt
att först undersöka om partiklarna är giftiga för celler eller aktiverar
immunförsvaret.
Ett immunsvar aktiveras av en akut inflammatorisk reaktion
som kan mätas via signalämnen som kallas cytokiner.
Vi mätte det
inflammatoriska svaret efter vi inkuberat immunceller med plastpartiklarna.
Resultaten visade ett svagt inflammatoriskt svar som var på samma nivå som
svaren som erhölls från två referenspartiklar.
Referenspartiklarna var partiklar
som idag används för medicinska ändamål. Dessutom analyserades om våra
partiklar var giftiga för immunceller efter 24-48 timmars inkubering med
partiklarna.
Våra resultat visade en minskad celltillväxt men inte någon ökad
celldöd.
Tillsammans visar detta att plastpartiklarna, inte är dödligt giftiga för
celler och att de inte orsakar et inflammatoriskt svar. Framtida arbete med
djurförsök är därför möjligt.
Sammanfattningsvis bidrar avhandlingens resultat till för att göra
cancerbehandling bättre och effektivare genom att ge ökad kunskap om
sialinsyra.
Avhandlingen bidrar också till utvecklingen av nya verktyg för
upptäckt av sialinsyra på celler, tidig diagnostik av cancer.
GlycoImaging
This work has been carried out within the framework of Marie Skłodowska-Curie
Actions European Training Network (ETN) “GlycoImaging: Imprinted sialic
acid nanoparticles for cancer associated biomarker detection” in 2017 - 2022.
The project has engaged a diverse team of chemists and biologists from five
research groups spread across five universities/institutes and two industrial
partners, distributed over four countries.
All with the same aim of developing
next generation tools for cancer research and diagnostics.
GlycoImaging is an interdisciplinary project that develops and implements highly
promising glycan specific probes for clinically relevant cancer diagnostic
technologies.
The project consists of five work packages focusing on separate
topics: synthesis of sialic acid molecularly imprinted polymers (SA-MIPs),
modified cell models, in vivo models, and cancer diagnostics.
This thesis has been
focused on digital holographic cytometry for evaluation of SA-MIPs targeting
cancer cells for ultimate in vivo applications.
The ETN has offered a great platform for collaborations between industrial and
academic partners, providing workshops and training events.
The molecularly
imprinted polymers were provided by the Federal Institute for Materials and
Research Testing (BAM), University of Copenhagen provided valuable insights
into glycomic workflows and gene-engineered cell lines, the University of Turku
and Umeå University assisted with cell staining and analysis, Malmö University
was in charge of cell-based studies on SA-MIPs.
Introduction
Alterations in glycosylation affect a wide spectrum of key biological processes
that are operational in development and progression of neoplastic diseases.
Tumor cells tend to induce the aberrant formation of glycoconjugates carrying
sialic acid (SA), thus establishing a negative charge to the effected glycan chains.
Being of nonbiological origin, engineered molecularly imprinted polymers
(MIPs), are extremely robust, resisting denaturing solvents and high
temperatures, and can be produced at low costs.
Having none of the limitations
of antibodies and lectins, MIPs have the potential to overcome many of the
problems of current detection strategies.
Detection of the morphology of cells and tissues by digital holographic cytometry
(DHC) is a long-term goal for researchers in the field.
Thus, cancer cells,
circulating tumor cells, and even metastatic cancer cells growing in in vivo
models can be screened using the DHC methodology.
Furthermore, even labelled
artificial receptors/MIP nanoparticles can be evaluated with DHC.
This could, in
turn, offer an instant clinical value.
This PhD project is a collaboration between the company Phase Holographic
Imaging (PHI) AB and Malmö University in the EU collaboration project
GlycoImaging.
This thesis deals with glycan-specific MIPs targeting cancer cells. The MIPs were
selective for SA (SA-MIPs), which we investigated regarding specificity for
cancer cells.
The DHC platform was used to study the morphology of cells, and
the possible role of SA in cancer cell movement.
Moreover, the cytotoxicity and
inflammatory response of SA-MIPs were analyzed to evaluate the possible use of
SA-MIPs in in vivo studies.
Digital holographic cytometry
In this thesis, the imaging technique digital holographic cytometry (DHC) is used
to obtain the main results presented in Paper II-IV, either isolated, or in
combination with other techniques.
The HoloMonitor M4 is a live cell time-lapse cytometer that employs digital
holography to allow non-invasive visualization and quantification of living cells
without compromising cell integrity.
The HoloMonitor M4 is based on a configuration called off-axis Mach-Zender
[93-95]. A 635 nm diode laser beam is split into two beams, the sample beam,
and the reference beam.
The sample beam passes through the sample, in our case,
the cells, before it is led to interfere with the reference beam (Figure 5). The
resulting interference pattern is captured on an image sensor and is used to create
a hologram by calculations based on the phase shift.
The phase shift is used to
calculate the actual thickness of the sample. A cell image can be constructed
based on the calculated cell thickness for every pixel of the image [90, 91].
There are
several advantages of holographic microscopy. One is that the created
quantitative phase images are focused when viewed rather than when recorded.
This makes the HoloMonitor M4 time-lapse cytometer ideal for long-term
imaging and analysis of living cells by means of time-lapse microscopy, where a
series of cell images are acquired at regular time intervals, allowing analysis of
the dynamics of various cellular events [100, 101].
Unfocused images, caused by
focus drift, are refocused by letting the computer software recreate the phase
image from the recorded hologram [102].
Another advantage of the HoloMonitor
M4 is that it can be installed in a cell incubator, and thereby it is possible to keep
the cells in the same environment as during normal cell culturing during the entire
experiment.
In Figure 6 below, a cell marked with a yellow border is shown at different time
points, and it is seen that the cellular morphology changes over time, in this
example, due to cell division.
With HoloMonitor M4 it is possible to both detect
these changes qualitatively and perform quantitative analysis by investigating
more than thirty different morphological parameters. In Figure 5e, some of the
most common morphological parameters are visualized. The first parameter is
the cell area.
The sample cell is first very large (Figure 5a), then it contracts and
becomes smaller (Figure 5b) and after cell division, each cell has a very small
area (Figure 5c); afterwards, the cell will flatten out again, and spread over a
larger area (Figure 5d). Initially, the optical thickness is relatively small, around
3.4 µm (Figure 5a). Preparing for cell division, the cell rounds up and becomes almost twice as thick (Figure 5b), and after cell division, it flattens out again
(Figure 5d). All the morphological parameters shift over time and when taken
together, the parameters tell a time-based story of the cell.
 |
| Figure 6. DHC images shows an example of cell division and how the cell changes in morphology when dividing using HoloMonitor proprietary software. a) a flatten out cell, b) a cell preparing for cell division, c) the cell has divided into two daugther cells, d) the cells are flatten out again, e) most common morphological parameters |
Conclusions
The evaluation of new tools for the detection of SA is important for the
development of future methods for cancer diagnosis. The research presented in
this thesis focuses on the binding of SA-MIPs to cancer cells and their
inflammatory and cytotoxic effects.
In addition, the study evaluated the
advantages of DHC, which is a state-of-the-art microscopy technique that
facilitates long term acquirements of quantitative data of living cells.
The major findings presented in Paper I-IV fulfills the aims of this thesis.
I. To investigate the binding behavior and specificity of SA-MIPs on
different cancer cell lines
✓ Different cancer cell lines have different and distinct SA expression
patterns and SA-MIPs generate similarly different binding patterns.
II. To study the potential of DHC by mapping cellular parameters
✓ DHC is a novel tool that can be used to discriminate different cell
types based on morphological alterations.
III. To investigate whether SA-MIPs lead to inflammatory and/or cytotoxic
responses when administered to phagocytosing cells
✓ The SA-MIPs had low inflammatory effect and expressed no shortterm cytotoxicity when administered to phagocytosing cells
In summary, these findings suggest that the synthesized SA-MIPs would be
applicable for future in vivo studies since they cause only minor in vitro
inflammatory responses as well as close to negligible cytotoxicity.
SA-MIPs have
the potential of becoming new tools for analysis of SAs in cancer diagnosis,
which is important for early detection and treatment of cancer, thus boosting the
survival rates of cancer patients.
Under Conclusions beskriver Louise även en viktig aspekt som förmodligen har bäring till den snart marknadserbjudna kombon Fluo/Holo.
Improving DHC
DHC analyses cells over time under stress free conditions, providing near-ideal
information about cell behavior. The drawback is that DHC lacks the ability to
identify cell surface markers such as proteins. Therefore, combining DHC with
fluorescence microscopy would facilitate additional identification of cells. One
main advantage would be the ability to match surface protein expression with cell
division and cell movement. Fluorescent imaging damages the cells, thus images
would have to be taken at a lower frequency, every 12 h interval, this would allow
unaffected cells growth.
DHC images can still be recorded in between
fluorescence captures to track individual cells.
*Min notering. DHC = Digital Holographic Cytometri (Holomonitor).
Avslutningsvis väljer bloggen att uppmärksamma delar där Louise nämner kliniskt arbete med DHC.
- Detection of the morphology of cells and tissues by digital holographic cytometry
(DHC) is a long-term goal for researchers in the field. Thus, cancer cells,
circulating tumor cells, and even metastatic cancer cells growing in in vivo
models can be screened using the DHC methodology.
Furthermore, even labelled
artificial receptors/MIP nanoparticles can be evaluated with DHC.
This could, in
turn, offer an instant clinical value.
DHC in the clinic
DHC is not only a powerful tool for research but could also serve as a useful tool
in the clinic. Classification of cells is a challenge that has attracted much
attention. DHC can classify leukocyte subpopulations based on their cellular size
and evaluate the morphological characteristic of erythrocytes.
This would aid the
diagnosis of diseases associated with erythrocytes e.g., malaria-infected
erythrocytes.
With DHC, the cellular shapes can be compared to determine
whether cells have been infected or not. Moreover, DHC has a wide spectrum of
applications in the biomedical field since it can perform high-throughput analyses
by monitoring and classifying various cell types.
It is an excellent technique for
long-term time-lapse imaging for determination of clinically relevant behavior of
cells. It has been demonstrated to be useful in for instance cervical cancer
screening, with the possibility of improving the screening process of gynecologic
cervical samples.
Allt strålkastarljus på snart titulerade Dr Louise Sternbæk.
Bloggen vill å PHI´s aktieägares vägnar utropa ett stort grattis i förskott till Louise.
Mvh the99
Inga kommentarer:
Skicka en kommentar